Regulation of function of angiopoietin

ABSTRACT

Pharmaceutical compositions that comprise a pharmaceutically acceptable carrier and either a therapeutically effective amount of an ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a homologous peptide thereof and pharmaceutical compositions that comprise a pharmaceutically acceptable carrier and a vector comprising a nucleic acid molecule that comprises the nucleotide sequence that encodes an ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a homologous peptide thereof are disclosed. Methods of using such compositions to treat individuals suspected of having coronary artery disease, vascular disease or a condition involving ischemia and to promote angiogenesis, endothelial survival and maintaining vascular integrity are disclosed. Methods to identify compounds that modulates binding of Ang-1 to ECM are disclosed. Pharmaceutical compositions which comprise a therapeutically effective amount of Ang-2 protein and/or a vector comprising a nucleic acid molecule that comprises the nucleotide coding sequence of Ang-2 and methods of using such compositions to treat individuals suspected of having cancer are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Ser. No. 60/359,679filed Feb. 26, 2002, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates activities of Angiopoietin-1 (Ang-1) andAngiopoietin-2 (Ang-2) and to uses of compounds having such activitiesto treat diseases and disorders and find additional compounds.

BACKGROUND OF THE INVENTION

Angiogenesis plays an important role in embryogenesis and tumorigenesis.It is a complicated multistep process, which includes the dynamicchanges of cell-cell and cell-matrix interactions, endothelial cellproliferation and migration, recruitment of the peri-vascular supportingcells, and the maturation process. Numerous molecules are involved inthose processes, including growth factors and their receptors,proteases, adhesion receptors, and the ECM1 components. VEGF andangiopoietin families play special roles in angiogenesis due to therestricted expression of their receptors.

Ang-1 and Ang-2 are ˜70 kDa with a considerable sequence homology, whichconsists of a signal peptide, an N-terminal coiled-coil domain, a shortlinker peptide region, and a C-terminal fibrinogen homology domain(FHD). The coiled-coil region is responsible fordimerization/multimerization of angiopoietins, and the fibrinogenhomology domain binds to Tie-2 receptor. Both Ang-1 and Ang-2 formdimers and oligomers.

Ang-1 and Ang-2 are the unique antagonists. Ang-1 induces tyrosinephosphorylation of Tie-2 receptor and promotes recruitment of thepericytes and smooth muscle cells, thereby playing a role inestablishing and maintaining the vascular integrity and quiescence. Asan antagonist of Ang-1, Ang-2 competes with Ang-1 for binding of Tie-2,blocks the phosphorylation of Tie-2 receptors induced by Ang-1, andloosens the interactions between endothelial and peri-vascular supportcells and ECM.

Targeted disruption of Ang-1 and Tie-2 and overexpression of Ang-2resulted in embryonic death with the similar vascular defects. Thosemice have normal primary vascular development, but the remodeling andmaturation of the vasculature are defective. The transgenic miceoverexpressing Ang-1 displayed increased vascularization and decreasedadult vasculature leakage. Together, those results indicated that Ang-1plays an indispensable role in the formation of blood vessels duringmouse development by recruiting and maintaining peri-endothelial supportcells.

Several studies have offered possible mechanisms for the pro-angiogeniceffect of Ang-1. Although Ang-1 does not stimulate the proliferation ofendothelial cells, it stimulates endothelial cell migration, induces thecapillary-like tubule formation, and promotes survival of endothelialcells. Ang-1 inhibits apoptosis of the endothelial cells viaphosphatidylinositol 3-kinase/Akt pathway.

Angiogenesis is regulated by the precise balance between pro- andanti-angiogenic factors. Ang-2 expression is often induced in theendothelia undergoing active remodeling or regression and by hypoxia andseveral growth factors, including VEGF. Ang-2 destabilizes thevasculature. Thus, it initiates angiogenesis in the presence of VEGF,which supplies endothelial cells with necessary survival andproliferation signals, or induces apoptosis of endothelial cells in theabsence of the pro-angiogenic factors.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to pharmaceuticalcompositions that comprise a pharmaceutically acceptable carrier andeither a therapeutically effective amount of an ECM-binding fragment ofAng-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or ahomologous peptide thereof.

Another aspect of the present invention relates to pharmaceuticalcompositions that comprise a pharmaceutically acceptable carrier and avector comprising a nucleic acid molecule that comprises the nucleotidesequence that encodes an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO:2 or a homologous peptidethereof.

A further aspect of the present invention provide methods of treating anindividual suspected of having coronary artery disease, vascular diseaseor a condition involving ischemia. In some embodiments, the methods compse the step of administering to the individual a pharmaceuticalcomposition that comprises a therapeutically effective amount of anECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1 and/orSEQ ID NO:2 or a homologous peptide thereof. In some embodiments, themethods comprise the step of administering to the individualpharmaceutical compositions that comprises a vector comprising a nucleicacid molecule that comprises the nucleotide sequence that encodes anECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1 and/orSEQ ID NO:2 or a homologous peptide thereof.

Another aspect of the invention provides methods of promotingangiogenesis, endothelial survival and maintaining vascular integrity inan individual. In some embodiments, the methods comprise the step ofadministering to the individual a pharmaceutical composition thatcomprises a therapeutically effective amount of an ECM-binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or ahomologous peptide thereof. In some embodiments, the methods comprisethe step of administering to the individual pharmaceutical compositionsthat comprises a vector comprising a nucleic acid molecule thatcomprises the nucleotide sequence that encodes an ECM-binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or ahomologous peptide thereof.

According to some other aspects of the invention, methods are providedto identify compounds that modulates binding of Ang-1 to ECM. Themethods comprise performing a test assay that comprises the steps ofcontacting a protein that comprises at least an ECM-binding fragment ofAng-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with ECMmaterial in the presence of a test compound, then measuring the level ofbinding of the protein that comprises at least an ECM-binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with theECM; and then comparing the level with the level of binding of proteinthat comprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO:2 with ECM material in theabsence of the test compound. When the level of binding of the proteinthat comprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO:2 with the ECM in the presence ofthe test compound is less than the level of binding of the protein thatcomprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO:2 with the ECM in the absence ofthe test compound results indicate that the test compound modulatesbinding of Ang-1 to ECM by inhibiting the binding. When the level ofbinding of the protein that comprises at least an ECM-binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with theECM in the presence of the test compound is more than the level ofbinding of the protein that comprises at least an ECM-binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with theECM in the absence of the test compound results indicate that the testcompound modulates binding of Ang-1 to ECM by enhancing the binding.

A further aspect of the invention provides pharmaceutical compositionswhich comprise a therapeutically effective amount of Ang-2 proteinand/or a vector comprising a nucleic acid molecule that comprises thenucleotide coding sequence of Ang-2.

Additional aspects of the invention provide for methods of treating anindividual suspected of having cancer. The methods comprise the step ofadministering to the individual a pharmaceutical composition comprisinga pharmaceutical composition which comprise a therapeutically effectiveamount of Ang-2 protein and/or a vector comprising a nucleic acidmolecule that comprises the nucleotide coding sequence of Ang-2.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1Aa, 1Ab, 1B and 1C disclose data related to angiopoietin-1incorporated into the extracellular matrix. The expression of Ang-1 andAng-2 in the serum-fee conditioned media (FIGS. 1Aa, 1Ab) and in the ECM(FIG. 1B) by the transfected LLC and TA3 cells were determined byWestern blot analyses using anti-v5 antibody (Invitrogen). The Westernblot analyses were performed under non-reducing conditions using theconcentrated serum-free media (FIGS. 1Aa, 1Ab) or the ECM extracts (FIG.1B) derived from two independent isolates of the transfected LLC cellsexpressing Ang-1v5 (FIG. 1Aa lanes 1 and 2, and FIG. 1B, lanes 1 and 2)or Ang-2v5 (FIG. 1Aa lanes 3 and 4, FIG. 1B lanes 3 ad 4); twoindependent isolates of the transfected TA3 cells expressing Ang-1v5(FIG. 1Ab lanes 1 and 2, FIG. 1B lanes 6 and 7) or Ang-2v5 (FIG. 1Ablanes 3 and 4, FIG. 1B lanes 8 and 9), and the tumor cells transfectedwith the expression vector only (LLC, carcinoma cells, FIG. 1Aa lane 5,FIG. 1B, lane 5; and TA3 cells, FIG. 1Ab lane 5, FIG. 1B, lane 10). FIG.1C, the same protein samples in the FIG. 1B were subjected to-mercaptoethanol (5%) treatment. Molecular mass markers are asindicated. kd indicates kilodalton.

FIG. 2 shows data related to the ECM association of angiopoietin-1. TheAng-1-containing ECM was extracted at room temperature for 10 min with0.15, 0.5, and 1 M NaCl and 0.5 and 1% DOC, respectively. The insolubleECM components were then extracted with 1×SDS Laemmli buffer andsubjected to Western blot analysis using anti-v5 antibody (lanes 2-6,respectively). The proteins in the lane 1 were derived from the ECMwithout prior extraction with any reagents. Lanes 7 and 8 are the ECMextracts derived from LLC cells expressing Ang-2 or transfected with theexpression vector alone, respectively. The molecular mass markers are asindicated.

FIGS. 3A and 3B show data demonstrating Ang-1 does not bind to heparin.In FIG. 3A, the purified v5-tagged Ang-1 proteins (500 ng/ml) wereloaded onto a heparin-Sepharose CL-6B affinity column. The flow-throughwas collected, and the column was washed with 0.15, 0.3, 0.5, and 1 Msodium chloride (NaCl, FIG. 3A, lanes 3-6), and the eluted proteins werecollected. All the collected fractions were subjected to Western blotanalysis using anti-v5 antibody. The results indicated that Ang-1v5 doesnot bind to heparin-Sepharose, and all the v5-tagged Ang-1 was in theflow-through fraction (FIG. 3A, lane 2). Lane 1 in FIG. 3A representsthe starting materials. In FIG. 3B, the ECM proteins were extracted, andWestern blot analysis was performed after the incubation of theconfluent LLC cells expressing Ang-1 with serum-free cell culture mediacontaining heparin (200 μg/ml, FIG. 3B, lane 2), chondroitin sulfate(200 μg/ml, FIG. 3B, lane 3), or SFM alone (FIG. 3B, lane 1) for 12 h.Molecular mass markers are as indicated.

FIG. 4 shows binding of Ang-1 to the different ECM components. Thebinding affinities of Ang-1 to several different ECM components weredetermined in the solid phase binding assays. Ang-1 exhibited noaffinity to fibronectin, laminin, collagen types I and IV (columns 1-4,respectively), heparin, chondroitin sulfate, and hyaluronic acid(columns 10 and 12, respectively). The weak affinities of Ang-1 tovitronectin and fibrinogen were observed (columns 5 and 6). Ang-1displayed a moderate affinity to Matrigel (column 7) and a strongaffinity to the 2 M urea ECM extracts derived from LLC carcinoma cells(column 8). All the experiments were performed in triplicate.

FIGS. 5Aa, 5Ab, 5Ac, 5Ad, 5Ba, 5Bb, 5Bc, 5Bd and 5Be show Ang-1displayed a distinct ECM distribution pattern. The distribution of Ang-1in LLC cells expressing Ang-1v5 was investigated by immunocytochemistryusing anti-v5 antibody (FIG. 5Aa). The distribution was compared withthat of fibronectin (FIG. 5Ba), laminin (FIG. 5Bb), collagen type I(FIG. 5Bc) and type IV (FIG. 5Bd), and Ang-2 in LLC cells expressingAng-2v5 (FIG. 5Ac). The cell-free ECM derived from the LLC carcinomacells expressing Ang-1 (FIG. 5Ab) or Ang-2 (FIG. 5Ad) was also analyzedby immunocytochemistry using anti-v5 antibody. LLC carcinoma cellsstained with the FITC-conjugated rabbit anti-mouse secondary antibodyonly are shown in FIG. 5Be. Bar, 40 μm.

FIG. 6 shows ECM-associated Ang-1 is released in the response to PMAstimulation. Several factors were tested for their abilities to releasethe ECM-sequestered Ang-1. After the incubation of the LLC carcinomacells expressing Ang-1 with SFM alone (lane 1) or SFM containing PMA(0.5 μg/ml, lane 2) TGF-1 (0.5 ng/ml, lane 3), bFGF (1 ng/ml, lane 4),epidermal growth factor (10 ng/ml, lane 5), heparin-binding epidermalgrowth factor (10 ng/ml, lane 6), and TGF-2 (0.5 ng/ml, lane 7),respectively, for 14 h, the cells were released from the cell culturedishes by the treatment of EDTA, and the remaining ECM components wereextracted with 1×SDS Laemmli buffer and subjected to the Western blotanalysis using anti-v5 antibody. Molecular mass markers are asindicated.

FIGS. 7A, 7Ba, 7Bb, 7Bc and 7Bd shows ECM-associated Ang-1 exhibited noaffinity to Tie-2-Fc fusion protein. A, Tie-2-Fc fusion protein iscapable of precipitating Ang-1 proteins from the protein extracts, whichwere derived from the 2 M urea extraction of the ECM of LLC carcinomacells expressing Ang-1 (A, lane 2). The starting materials for theimmunoprecipitation were loaded in lane 1 (A), and the control for theimmunoprecipitation contains everything except Tie-2-Fc fusion proteins(A, lane 3). Molecular mass markers are as indicated. B, the LLC cellsexpressing Ang-1 or the ECM derived from LLC carcinoma cells expressingAng-1 were incubated with Tie-2-Fc fusion proteins (B, c and d) oranti-v5 antibody (B, a and b), and FITC-conjugated rabbit anti-human Fcor anti-mouse secondary antibodies were used, respectively. Bar, 30 μm.

FIGS. 8A and 8B show Tie-2 receptors on HUVECs phosphorylated upon theadherence of HUVECs to the Ang-1-containing ECM. The serum-starvedHUVECs were lifted by the treatment of EDTA/Hanks' balanced saltsolution, and 1×106 of the HUVECs were seeded into the plastic dishcontaining SFM with (200 ng/ml, lane 3) or without soluble Ang-1(lane 1) or the dishes coated with the ECM derived from LLC cellsexpressing Ang-2 (lane 2) or Ang-1 in the presence (lane 5, 2 μg/ml) orabsence of Tie-2-Fc fusion proteins (lane 4) for 30 min. The HUVECs werelysed, and 50 μg of proteins from each lysate were subjected to Westernblot analysis using anti-Tie-2 antibody (Santa Cruz Biotechnology, FIG.8B). The rest of the proteins were used in the immunoprecipitation usinganti-Tie-2 antibody (Santa Cruz Biotechnology). The immunoprecipitatedproteins were subjected to Western blot analysis usinganti-phosphotyrosine (FIG. 8A). Molecular mass markers are as indicated.

FIGS. 9Aa, 9Ab, 9Ac, 9Ad, 9B and 9C show the linker peptide region ofAng-1 is responsible for its ECM association. Several cDNA expressionconstructs were made including the full-length of Ang-1 or Ang-2, thecoiled-coil region, the coiled-coil plus the linker peptide region, thefibrinogen-like region of Ang-1 (FIGS. 9Aa, 9Ab, 9Ac, 9Ad). All theAng-1 fragments contain N-terminal signal peptides of Ang-1 and theC-terminal v5 epitope tags. These expression constructs were used totransfect COS-7 cells transiently. 72 h after the transfection, the cellculture supernatants (FIG. 9B) and the ECM proteins (FIG. 9C) werecollected or extracted and subjected to Western blot analyses todetermine the distribution of Ang-1, Ang-2, and the Ang-1 fragmentsusing anti-v5 antibody. Lanes 1 and 2, full-length Ang-1; lanes 3 and 4,full-length Ang-2; lanes 5 and 6, the coiled-coil domain plus the linkerpeptide region of Ang-1; lanes 7 ad 8, the coiled and coil region ofAng-1; lanes 9 and 10, the fibrinogen homology domain of Ang-1; lane 11,COS-7 cells transfected with the expression vector alone. Molecular massmarkers are as indicated.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, the term “ECM” refers to the extracellular matrix. Theextracellular matrix (ECM) is a complex structural entity surroundingand supporting cells that are found within mammalian tissues. The EOM iscomprises, structural proteins (collagen and elastin), specializedproteins (e.g. fibrillin, fibronectin, and laminin), and proteoglycans.Proteoglycans are composed of a protein core to which is attached longchains of repeating disaccharide units termed of glycosaminoglycans(GAGs) forming extremely complex high molecular weight components of theECM.

The present invention relates to peptides and methods using thosepeptides to treat an individual suspected of having cancer, coronaryartery disease, ischemias, and other vascular diseases. Although thespecific procedures and methods described herein are exemplified usingseveral specific peptides derived from Angiopoietin-1 and -2, they aremerely illustrative for the practice of the invention. Analogousprocedures and techniques, as well as functionally equivalent peptidesand peptide homologues, as will be apparent to those of skill in the artbased on the detailed disclosure provided herein are also encompassed bythe invention.

Aspects of the present invention arises from the discovery thatangiopoietin-1 (Ang-1) associates with extracellular matrix (ECM), andthe function of Ang-1 is regulated by the association with the ECM.Thus, while Ang-1 promotes angiogenesis when not associated with theECM, it is inhibited from promoting angiogenesis while associated withthe ECM.

According to some embodiments the present invention provides forpharmaceutical compositions comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of an ECM-bindingfragment of the Ang-1 protein and/or a vector comprising a nucleic acidmolecule that comprises the nucleotide sequence that encodes anECM-binding fragment of Ang-1 protein.

As used herein, the term “ECM-binding fragment of Ang-1 protein” refersto any peptide sequence that comprises a peptide fragment from Ang-1that can bind to the ECM. In some embodiments the ECM-binding fragmentof Ang-1 protein is the 26-mer peptide VHNLVSLCTKEGVLLKGGKREEEKPF (SEQID NO:1). In some embodiments the ECM binding fragment of Ang-1 proteinis VHNLVNLCTKEGVLLKGGKREEEKPF (SEQ ID NO:2). The fragment may be theentire Ang-1 protein or it may be a fragment of the Ang-1 protein. Insome embodiments, the ECM-binding fragment of Ang-1 protein may be apart of a fusion protein that comprises Ang-1 protein sequence andnon-Ang-1 protein sequence. In some embodiments the ECM-binding fragmentof Ang-1 protein is at least 26, at least 50, at least 75, at least 100,at least 500, at least 1000 amino acid residues long. In someembodiments the ECM-binding fragment of Ang-1 protein comprises SEQ IDNO:4, SEQ ID NO:6, and/or SEQ ID NO:8

As used herein, the term “homologous peptide” refers to a peptide thathas at least 50% similarity to the peptide being referred to. In someembodiments the peptide has at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 100% similarity to the ECM-binding fragment ofAng-1 protein and can bind to the ECM. In some embodiments thehomologous peptide has at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 100% similarity to SEQ ID NO:1, SEQ ID NOs:2, SEQID NO:4, SEQ ID NO:6, and/or SEQ ID NO:8. The homologous peptide may beisolated or incorporated into another protein so that a fusion proteinis created.

According to some embodiments a homologous peptide refers to a peptidethat has conservative substitutions. A conservative substitution isrecognized in the art as a substitution of one amino acid for anotheramino acid that has similar properties. In some embodiments examples ofconservative substitutions are those that are described in Table I.

TABLE I Amino Acid Conservative Changes Alanine (A) Glycine (G), Serine(S) Aspartic Acid (D) Glutamic Acid (E) Glutamic Acid (E) Aspartic Acid(D) Phenylalanine (F) Tryptophan (W), Tyrosine (Y) Glycine (G) Alanine(A) Histidine (H) Tyrosine (Y) Isoleucine (I) Leucine (L), Methionine(M), Valine (V) Lysine (K) Arginine (R) Leucine (L) Isoleucine (I),Methionine (M) Valine (V) Methionine (M) Isoleucine (I), Leucine (L),Valine (V) Asparagine (N) Glutamine (Q) Glutamine (Q) Asparagine (N)Arginine (R) Lysine (K) Serine (S) Alanine (A), Threonine (T) Threonine(T) Serine (S) Valine (V) Isoleucine (I), Methionine (M) Valine (V)Tryptophan (W) Phenylalanine (F), Tyrosine (Y) Tyrosine (Y)Phenylalanine (F) Histidine (H) Tryptophan (W)

As used herein, the phrase “homologous”, “homologous peptide”,“homologous peptide thereof” or variations thereof, refers to sequencescharacterized by a homology, at the nucleotide level or amino acidlevel, of at least a specified percentage. Homologous nucleotidesequences include those sequences coding for isoforms of proteins. Suchisoforms can be expressed in different tissues of the same organism as aresult of, for example, alternative splicing of RNA. Alternatively,isoforms can be encoded by different genes. Homologous nucleotidesequences include nucleotide sequences encoding for a protein of aspecies other than humans, including, but not limited to, mammals.Homologous nucleotide sequences also include, but are not limited to,naturally occurring allelic variations and mutations of the nucleotidesequences set forth herein. Homologous amino acid sequences includethose amino acid sequences which contain conservative amino acidsubstitutions and which polypeptides have the same binding and/oractivity.

Percent homology, similarity, or identity can be determined by, forexample, the Gap program (Wisconsin Sequence Analysis Package, Versionfor Unix, Genetics Computer Group, University Research Park, MadisonWis.), using default settings, which uses the algorithm of Smith adWaterman (Adv. Appl. Math., 1981, 2, 482-489). In some preferredembodiments, homology between the probe and target is between about 50%to about 60%. In some embodiments, nucleic acids have nucleotides thatare about 60%, preferably about 70%, more preferably about 80%, morepreferably about 85%, more preferably about 90%, more preferably about92%, more preferably about 94%, more preferably about 95%, morepreferably about 97%, more preferably about 98%, more preferably about99% ad most preferably about 100% homologous to nucleotide sequencesdisclosed herein.

Homology may also be at the polypeptide level. In some embodiments,polypeptides are about 50%, about 60%, preferably about 70%, morepreferably about 80%, more preferably about 85%, more preferably about90%, more preferably about 92%, more preferably about 94%, morepreferably about 95%, more preferably about 97%, more preferably about98%, more preferably about 99% and most preferably about 100% homologousto the polypeptide sequences disclosed herein.

As used herein, the term “fusion protein” refers to a protein thatcomprises amino acids that are from at least two different proteins. Asan example, a fusion protein may comprise the ECM-binding fragment ofAng-1 protein and kinase domain of another protein. This example wouldbe considered a fusion protein. The fusion of two protein sequences canbe in any orientation. The ECM-binding fragment may be placed at theN-terminus of a fusion protein or at the C-terminus of a fusion protein.In some embodiments, the ECM-binding fragment of Ang-1 protein may beplaced in the middle of a protein.

As used herein, the term “homologous peptide thereof” refers to apeptide that is a homologous peptide, as defined above, to theECM-binding fragment of Ang-1 protein. The homologous peptide thereofmay be fusion protein, the entire Ang-1 protein, or a fragment thereof.

As used herein, the term “nucleotide sequence that encodes anECM-binding fragment of Ang-1 protein” refers to a nucleotide sequencethat when transcribed and translated would comprise an ECM-blindingfragment of Ang-1 protein. According to some embodiments of the presentinvention, the nucleotide sequence may be the entire sequence of Ang-1.In some embodiments the nucleotide sequence may comprise a fragment ofthe nucleotide sequence of Ang-1. In some embodiments the nucleotidesequence that encodes an ECM-binding fragment of Ang-1 protein comprisesSEQ ID NOs: 3, 5, and/or 7. The nucleotide sequence of Ang-1 is wellknown to one of ordinary skill in the art.

As used herein, the term “pharmaceutical composition” refers tocompositions according to the invention including delivery components incombination with nucleic acid molecules and/or peptide molecules whichfurther comprise a pharmaceutically acceptable carriers or vehicles,such as, for example, saline. Any medium may be used which allows forsuccessful delivery of the peptide and/or nucleic acid. One skilled inthe art would readily comprehend the multitude of pharmaceuticallyacceptable media that may be used in the present invention.

Pharmaceutical compositions may be formulated by one having ordinaryskill in the art with compositions selected depending upon the chosenmode of administration. Suitable pharmaceutical carriers are describedin Remington's Pharmaceutical Sciences, A. Osol, a standard referencetext in this field, which is incorporated herein by reference.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the active agent to reach theagent's site of action in the body of an individual. Pharmaceuticalcompositions may be administered parenterally, i.e., intratumor,intravenous, subcutaneous, intramuscular. Intravenous and intratumoradministration are preferred routes. Dosage varies depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired.

As used herein, the term “vector” refers to a delivery vehicle that iscapable of delivering a nucleic acid to a cell. In some embodiments, thevector is a viral vector. In general, viral vectors may be DNA virusessuch as recombinant adenoviruses and recombinant vaccinia viruses or RNAviruses such as recombinant retroviruses. Other recombinant vectorsinclude recombinant prokaryotes that can infect cells and expressrecombinant genes. In addition to recombinant vectors, other vectors arealso contemplated such as encapsulation in liposomes,lipofectin-mediated transfection, transferrin-mediated transfection andother receptor-mediated means. In some embodiments the vector is a DNAplasmid. The invention is intended to include such other forms ofexpression vectors and other suitable delivery means which serveequivalent functions and which become known in the at subsequentlyhereto.

Examples of recombinant adenoviral vectors include those which have theE1a region deleted and which carry a temperature-sensitive mutation inE2a (Engelhardt et al., Hum Gene Ther 5:1217-1229, 1994, which isincorporated herein by reference). Other examples of recombinantadenoviral vectors useful to deliver nucleic acid sequence of thepresent invention are described in U.S. Pat. Nos. 5,756,283 and5,707,618, which are each incorporated herein by reference.

In another preferred embodiment of the present invention, RNA isdelivered to competent host cells by means of a retrovirus. One skilledin the art would readily understand this technique of delivering RNA toa host cell by such means. Any retrovirus which serves to express theprotein encoded by the RNA is intended to be included in the presentinvention.

In another preferred embodiment of the present invention, nucleic acidis delivered through folate receptor means. The nucleic acid sequence tobe delivered to a host cell is linked to polylysine and the complex isdelivered to the tumor cell by means of the folate receptor. U.S. Pat.No. 5,108,921 issued Apr. 28, 1992 to Low et al., which is incorporatedherein by reference, describes such delivery components.

In another preferred embodiment of the present invention, nucleic acidis delivered through the use of lipofectin-mediated DNA transfer.LipofectAMINE™ liposome reagent (Life Technologies, Gaithersburg Md.) isa commercially available liposome encapsulation reagent which can beused for encapsulating cells following manufacturer's instructions.LipofectAMINE™ liposome reagent encapsulated nucleic acid molecules maybe delivered to a host cell using liposome formulation administrationmethods.

In another preferred embodiment of the present invention, nucleic acidis delivered through the use of cationic lipid-mediated DNA transfersuch as that which is described in U.S. Pat. No. 5,703,055, which isincorporated herein by reference.

In another preferred embodiment of the present invention, nucleic acidis delivered through the use of liposome-mediated DNA transfer such asthat which is described in U.S. Pat. Nos. 4,235,871, 4,241,046 and4,394,448, which are each incorporated herein by reference.

According to some embodiments the present invention provides methods oftreating an individual suspected of having coronary artery disease,vascular disease, or a condition involving ischemia. In some embodimentsthe method comprises the steps of administering to the individual apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of an ECM-bindingfragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2or a homologous peptide thereof and/or a vector comprising a nucleicacid molecule that comprises the nucleotide sequence that encodes anECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1 and/orSEQ ID NO:2 or a homologous peptide thereof. In some embodiments, themethods are provided for individuals need thereof.

As used herein, the term “coronary artery disease” refers to diseasesthat are a result of the buildup of cholesterol in the inside layers ofthe arteries. As used herein, the term “vascular disease” refers todiseases that is related to the circulatory system. As used herein, theterm “ischemia” refers to a condition that is a caused by a lack ofblood flow that would otherwise be present in a healthy individual.

In some embodiments the compounds of the invention, may be administeredto a subject per se or in the form of a pharmaceutical composition.Pharmaceutical compositions comprising the compounds of the inventionmay be manufactured by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the active peptides or peptide analogues intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For topical administration the compounds of the invention may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Alternatively, the compounds may be in powder form for constitution witha suitable vehicle, e.g., sterile pyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be readily formulated bycombining the active peptides or peptide analogues with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompounds of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a patient to be treated. For oral solid formulationssuch as, for example, powders, capsules and tablets, suitable excipientsinclude fillers such as sugars, such as lactose, sucrose, mannitol andsorbitol; cellulose preparations such as maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP); granulating agents; and binding agents. Ifdesired, disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

If desired, solid dosage forms may be sugar-coated or enteric-coatedusing standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

For buccal administration, the compounds may take the form of tablets,lozenges, etc. formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g, containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat may be used to deliver peptides and/or nucleotides of theinvention. Certain organic solvents such as dimethylsulfoxide also maybe employed, although usually at the cost of greater toxicity.Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

According to some embodiments of the present invention thepharmaceutical composition is administered in an amount that istherapeutically effective. As used herein, the term “therapeuticallyeffective” refers to an amount effective to achieve the intendedpurpose. In some embodiments the intended purpose is to treat coronaryartery disease, vascular disease, a condition involving ischemia, orcancer. In some embodiments a therapeutically effective amount refers toan amount effective to ameliorate or prevent the symptoms, or prolongthe survival of the patient being treated. Therapeutically effectiveamounts are typically determined by the effect they have compared to theeffect observed when a composition which includes no active ingredientis administered to a similarly situated individual The precise effectiveamount for a subject will depend upon the subject's size and health, thenature and extent of the condition, and the therapeutics or combinationof therapeutics selected for administration. However, the effectiveamount for a given situation is determined by routine experimentationand is within the judgment of the clinician. Determination of atherapeutically effective amount is well within the capabilities ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the compounds which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5to 1 mg/kg/day. Therapeutically effective serum levels may be achievedby administering multiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of the compounds may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

The amount of compound administered will, of course, be dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable. The therapy may be provided alone orin combination with other drugs.

According to some embodiments, the present invention provides formethods of promoting angiogenesis, endothelial survival, and/ormaintaining vascular integrity comprising the administration of apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of an ECM-bindingfragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2or a homologous peptide thereof and/or a vector comprising a nucleicacid molecule that comprises the nucleotide sequence that encodes anECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1 and/orSEQ ID NO:2 or a homologous peptide thereof.

As used herein, the term “angiogenesis” refers to the growth of bloodvessels. In some embodiments the promotion of angiogenesis promotes thegrowth of new blood vessels, while in some embodiments existing bloodvessels are promoted to grow. Angiogenesis is a term well understood bythose of ordinary skill in the art. In some embodiments endothelialsurvival refers to the process of preventing endothelial cells fromdying. In some embodiments endothelial survival refers to the promotingthe growth of endothelial cells.

In some embodiments “maintaining vascular integrity” refers to theprocess by which the a vascular system viability and functions are keptat specific level. In some embodiments the vascular system may belocated throughout the individual. In some embodiments the vascularsystem may be localized to a specific region of the individual. Forexample, if a person has a poor vascular system in the foot, thepharmaceutical composition may be administered in a therapeuticallyeffective amount to promote and maintain vascular integrity in thatfoot, while the rest of the vascular system may be unaffected. However,in other embodiments a therapeutically effective amount may promoteangiogenesis, endothelial survival, and maintaining vascular integritythroughout the individual.

According to some embodiments, the present invention provides formethods for identifying compounds that modulates the binding of Ang-1 toECM comprising performing a test assay that comprises the steps ofcontacting a protein that comprises at least an ECM-binding fragment ofAng-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with ECMmaterial in the presence of a test compound and measuring the level ofbinding of the protein that comprises at least an ECM binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with theECM. In some embodiments the method further comprises comparing thelevel with the level of binding of the protein that comprises at leastan ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1and/or SEQ ID NO:2 with ECM material in the absence of said testcompound. In some embodiments when the level of binding of the proteinthat comprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and SEQ ID NO:2 with the ECM in the presence ofthe test compound is less than the level of binding of the protein thatcomprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO: 2 with the ECM in the absence ofthe test compound results indicate that the test compound modulatesbinding of Ang-1 to ECM by inhibiting the binding.

In some embodiments when the level of binding of the protein thatcomprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO: 2 with the ECM in the presenceof the test compound is more than the level of binding of the proteinthat comprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO: 2 with the ECM in the absence ofthe test compound results indicate that the test compound modulatesbinding of Ang-1 to ECM by enhancing the binding.

As used herein, the term “modulates” refers to an increase or adecrease. In some embodiments the test compound increases the level ofAng-1 protein binding to the ECM. In some embodiments the test compounddecreases the level of Ang-1 protein binding to the ECM.

As used herein, the term “at least an ECM-binding fragment of Ang-1protein” refers to a protein that comprises a fragment of Ang-1 that canbind to ECM. In some embodiments this refers to SEQ ID NO:1 and/or SEQID NO:2. In other embodiments, this refers to protein that comprises asection of the protein that is homologous to an ECM-binding fragment ofAng-1 protein. In some embodiments, the protein can be the full-lengthAng-1 protein or a fragment thereof. In some embodiments the “at leastan ECM-binding fragment of Ang-1 protein” comprises SEQ ID NO:4, SEQ IDNO:6, and/or SEQ ID NO:8. In some embodiments, the protein can be afusion protein that comprises Ang-1 protein sequence and non-Ang-1protein sequence. In some embodiments, a protein comprising at least anECM-binding fragment of Ang-1 protein comprises a peptide sequence thathas at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 100% similarity to an ECM-binding fragment of Ang-1. Insome embodiments a protein comprising at least an ECM-binding fragmentof Ang-1 protein comprises a peptide sequence that has at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 100%similarity to SEQ ID NO:4, SEQ ID NO:6, and/or SEQ ID NO:8. In someembodiments a protein comprising at least an ECM-binding fragment ofAng-1 protein comprises a peptide sequence that has at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 100%similarity to SEQ ID NO:1 and/or SEQ ID NO:2.

According to some embodiments “ECM material” refers to a compound ormaterial that is found in the extracellular matrix that can bind to anECM-binding fragment of Ang-1 protein. According to some embodiments the“ECM material” refers to a composition comprising fibronection, laminin,type I collagen, type IV collagen, vitronectin, fibrinogen, matrigel,LLC carcinoma ECM, BSA, heparin, chondroitin sulfate, or hyaluronicacid.

In some embodiments the ECM material is produced by culturing cells on asubstrate for a sufficient time for the cells to produce the ECMmaterial on the substrate and then removing the cells from the substratewithout removing the ECM material. In some embodiments the cells thatare used to produce the ECM material are Lewis Lung carcinoma cells orTA3 murine mammary carcinoma cells. As used herein, the term “substrate”refers to any vessel or container that is capable of culturing cells.Examples of substrates include, but are not limited to, petri dishes,6-well plates, 96-well plates, 384-well plates, and the like. Removingcells from the substrate without removing the ECM material is wellwithin the skill of one of ordinary skill in the art. An example of howto remove the cells without removing the ECM includes contacting thecells with a chelator such as EDTA or EGTA for a sufficient time toremove the cells without effecting the ECM material. There are othermethods that can performed the same function as contacting the cellswith EDTA or EGTA and are within the scope of the current invention.

According to some embodiments the protein that comprises at least anECM-binding fragment of Ang-1 comprising a detectable label. As usedherein, the term “detectable label” refers to any molecule that can bedetected with methods that are well known to those of ordinary skill inthe art. Molecules with detectable labels include without limitationproteins, protein fragments, antibodies, fluorescent labels, radioactivelabels, chromophores, chemiluminescent probes, and the like. In someembodiments the detectable label is used to measure the level of bindingof the protein that comprises at least an ECM-binding fragment of Ang-1protein on the ECM.

In some embodiments the method of identifying compound that modulatesthe binding of Ang-1 to ECM further comprises multiple test assays thatare identical except that the amount of the test compound used differs.To aid in determining the effective amount of a test agent multipleassays are preformed using different amounts of the test compound. Insome embodiments at least 2 assays are performed. In some otherembodiments at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 15, at least 20, at least30, at least 40, at least 50, at least 100 assays are performed usingdifferent amounts of the test compound.

According to some embodiments the methods of identifying a compound thatmodulates the binding of Ang-1 to ECM further comprises determining thelevel of binding of a protein that comprises at least an ECM-bindingfragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2with ECM material in the absence of the test compound by performing acontrol assay wherein the control assay comprises the steps ofcontacting a protein that comprises at least an ECM-binding fragment ofAng-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with ECMmaterial in the absence of a test compound and measuring the level ofbinding of the protein that comprises at least an ECM binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with theECM.

Assays that can be used for the methods to identify compounds thatmodulates the binding of Ang-1 to ECM are well known to those ofordinary skill in the art and require only routine experimentationExamples of assays that are well known to those of ordinary skill in theart include ELISA, Sandwich Assays, flow cytometry, immunoprecipitation,and the like.

According to some embodiments the present invention provides forpharmaceutical compositions comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of Ang-2 protein and/or avector comprising a nucleic acid molecule that comprises the nucleotidecoding sequence of Ang-2.

As used herein, the term “Ang-2” refers to the protein or nucleic acidencoding the protein or fragment thereof of Angiopoietin-2. In someembodiments the Ang-2 is mammalian Ang-2. In some embodiments, the Ang-2is human, mouse, rat, dog, cat, pig, or horse. In some embodiments theAng-2 protein comprises SEQ ID NO:10 and/or SEQ ID NO:12. In someembodiments the Ang-2 nucleotide coding sequence comprises SEQ ID NO 9and/or SEQ ID NO:11. In some embodiments, the Ang-2 protein or thenucleic acid that encodes Ang-2 is a fragment of the Ang-2 protein orthe nucleotide coding sequence of Ang-2. In some embodiments the Ang-2protein comprises a fragment of SEQ ID NO:10 and/or SEQ ID NO:12. Insome embodiments, “Ang-2” refers to a fusion protein comprisingnon-Ang-2 protein sequence and Ang-2 protein sequence. According to someembodiments, “Ang-2” refers to a protein that has at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 100% similarityto SEQ ID NO:10 and/or SEQ ID NO:12. In some embodiments the nucleotidecoding sequence comprises a nucleotide coding sequence that is at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least100% identical to SEQ ID NO:9 and/or SEQ ID NO:11.

According to some embodiments the present invention provides for methodsof treating an individual, or an individual in need thereof, suspectedof having cancer comprising the step of administering to the individuala pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of Ang-2 protein and/or avector comprising a nucleic acid molecule that comprises the nucleotidecoding sequence of Ang-2. The methods of administration are definedabove as well as what is meant by a therapeutically effective amount.

The cancers that can be treated are not limited to any cancer describedherein and can include cancers of the bladder, cancers of the brain,cancers of the breast, cancers of the colon, hodgkin's disease, cancersof the kidney, cancers of the lung, melanoma, non-hodgkin's lymphoma,oral cancer, ovarian cancer, prostate cancer, uterine/cervical cancer,leukemia, cancers of the pancreas, testicular cancer, solid tumors, andthe like.

Examples

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) affect angiogenesisdifferently during embryogenesis and tumorigenesis. In an attempt tounderstand the molecular basis underlying the distinct roles of thosetwo homologous molecules, we investigated the association of Ang-1 andAng-2 with the extracellular matrix (ECM). TA3 murine mammary carcinoma(TA3) and Lewis lung carcinoma cells expressing v5 epitope-tagged Ang-1and Ang-2 were used in our studies. The results indicated that Ang-1 issecreted and incorporated into the ECM of the tumor cells, whereas Ang-2is not associated with the ECM. The mutagenesis study indicated thedomain that is responsible for the ECM association of Ang-1 is thelinker peptide region between the coiled-coil and the fibrinogen-likedomains. A weak binding between the coiled-coil domain of Ang-1 and theECM was observed. Immunocytochemistry study revealed a distinct ECMdistribution pattern of Ang-1, which is quite different from that offibronectin, laminin, and collagen types I and IV. The ECM-associatedAng-1 proteins are released, and Tie-2 receptors are phosphorylated uponthe adhesion of human umbilical vein endothelial cells. Implications ofthe difference in the ECM association of Ang-1 and Ang-2, which arerelated to the regulation of angiopoietin activity and their roles inlocal versus distant angiogenesis during tumor metastasis, arediscussed.

Ang-1 and -2 are expressed by tumor cells. We found that overexpressionof exogenous Ang-2, but not Ang-1, inhibits growth and metastasis ofLewis lung carcinoma (LLC) and TA3 murine mammary carcinoma cells. Thetumors overexpressing Ang-2 exhibited aberrant and incompleteangiogenesis in vivo, which is characterized by formation of thedisorganized endothelial cell aggregates, the lack of endothelialassociated smooth muscle cells, and massive apoptosis of the endothelialcells and surrounding tumor cells. This result is consistent with thenotion that Ang-2 inhibits the Ang-1-dependent recruitment of smoothmuscle cells.

To reveal the molecular basis underlying the different roles ofangiopoietins in tumor angiogenesis, we investigated the relationshipbetween angiopoietins and the ECM in the present study. We found that,unlike Ang-2, Ang-1 is secreted and incorporated into the ECM via itslinker peptide region. The association between Ang-1 and the ECM isstrong, and the distribution of Ang-1 in the ECM is unique and differentfrom that of fibronectin, laminin, and collagen type I and type IV. Thereleasing or incorporation of Ang-1 from or into the ECM is regulated bydifferent factors. Tie-2 phosphorylation was detected in HUVECs seededonto the ECM containing Ang-1, which indicates a regulatory role of theECM association of Ang-1 in tumor angiogenesis.

Ang-2 expression is regulated by hypoxia and growth factors. UnlikeAng-2, little is known about the regulation of Ang-1 expression. Thefinding reported herein offers a possible regulatory mechanism for theavailability of Ang-1 proteins, that is instead of regulating theproduction of Ang-1, it may be regulated by its ECM association. Thestrong ECM binding of Ang-1 implies the effect of Ang-1 is limited tothe local environment where it is produced, whereas Ang-2 can diffuse toand affect angiogenesis in the distant sites.

Experimental Procedures

Cell Culture and Reagents—Lewis lung carcinoma (LLC, ATCC), TA3 mammarycarcinoma (37) cells, and the tumor cell transfectants were maintainedas described (17). Anti-laminin, -fibronectin (Sigma), -collagen types Iand IV (Biodesign international), -phosphotyrosine (PY20, Calbiochem),-Tie-2 (C-20, Santa Cruz Biotechnology), -Ang-1 (C-19, Santa CruzBiotechnology), and -v5 antibodies were used.

Construction of the Expression Vectors—Full-length Ang-1 and Ang-2 cDNAswere generated by RT-PCR as described (17, 40). The coiled-coil domainand the coiled-coil plus the linker peptide region were generated byRT-PCR using the following pairs of primers. The forward primer for thecoiled-coil domain is 5′-ACAATGACAGTTTTCCTTTCCTTT-3′ and the reverseprimer is 5′-TGTGTCCATGAGCTCCAGTTGTTG-3′. The coiled-coil plus thelinker peptide region was amplified using the same forward primer asthat of the coiled-coil domain and a reverse primer,5′-AAATGGTTTCTCTTCTTCTCTTTT-3′. The stop codons were omitted from thereverse primers so that the v5 and 6× histidine tags in the pEF6/V5-Hisexpression vector (Invitrogen) can be attached to the C terminus of theAng-1 fragments. To fuse the FHD of Ang-1 to the signal peptide ofAng-1, we used the full-length Ang-1 in the expression vector (17) as atemplate and the ExSite PCR-based site-directed mutagenesis kit(Stratagene) together with a forward primer derived from the beginningof the FHD, 5′-CGAGACTGTGCAGATGTATATCAA-3′, and a reverse primer derivedfrom the end of the signal peptide, 5′-TCTTCTCCCTCCGTTTTCTGGATT-3′. Theextracellular domain of Tie-2 was obtained by RT-PCR using the followingpair of primers. The forward primer is 5′-AGTATGGACTCTTTAGCCGGCTTA-3′and the reverse primer is 5′-CATCTTTCCCCCTCCGAGGTCTGC-3′. The PCRproducts were inserted in frame into 5′-end of the Fc fragments of humanIgG in pEF6/V5-His expression vectors to generate the Tie-2-Fc fusionconstruct (40). The authenticity and orientation of the cDNA insertswere confirmed by DNA sequencing.

Transfection—LLC and TA3 carcinoma cells were transfected with theexpression constructs containing Ang-1, Ang-2, or the expression vectoralone. The transfected cells expressing Ang-1 and Ang-2 were identifiedas described (17). COS-7 cells were used in the transient transfectionof Ang-1 and -2, the coiled-coil domain, the coiled-coil plus linkerregion, and the FHD of Ang-1 using LipofectAMINE (Life Technologies,Inc.) as described (17).

Preparation of the Secreted and the ECM-Associated Proteins and WesternBlot Analysis—The cell culture supernatants of LLC and TA3 transfectantsor the transiently transfected COS-7 cells were collected. The confluentcells layers were released from the culture dishes by incubating withPBS containing 5 mM EDTA. The ECM components remaining on the culturedishes were washed and extracted with 1×SDS Laemmli sample buffer withor without 5%-mercaptoethanol.

To determine the affinity of the ECM association of Ang-1, the confluentLLC cells expressing Ang-1 were lifted as described. The remaining ECMcomponents were extracted with 0.5 M PBS and 1 M sodium chloride (NaCl)and 0.5 and 1% deoxycholate (DOC) at room temperature for 10 min. Theremaining insoluble materials were solubilized by 1×SDS Laemmli samplebuffer and subjected to Western blot analysis.

Immunocytochemistry—LLC cells expressing Ang-1 or Ang-2 or transfectedwith the expression vectors were cultured in 35-mm dishes untilsubconfluence or confluence. They were fixed with methanol at 20° C. for15 min. Antibodies against v5 epitope, fibronectin, laminin, type I orIV collagens were used to detected Ang-1v5, Ang-2v5, fibronectin,laminin, types I or IV collagen, respectively, in those fixed cells.

Extraction of the ECM Components and Affinity Purification—The ECMmaterials derived from the cultured LLC cells were extracted overnightat 4° C. in 2 M urea and 0.05 M Tris-HCl (pH 7.4). The soluble materialswere dialyzed against PBS and used to coat the enzyme-linkedimmunosorbent assay plates. The ECM derived from the LLC transfectantsexpressing Ang-1 was extracted in the same way. The ECM extractscontaining Ang-1 proteins and the serum-free culture media containingsecreted Ang-2, both of which contain v5 and 6× histidine tags at theirC-terminal ends, were loaded onto Ni+ Probond affinity columns(Invitrogen) and purified following the manufacturer's instructions.

Purified Ang-1v5/His proteins were loaded onto a heparin-Sepharose CL-6Bcolumn to test their affinity to heparin. The flow-through fraction wascollected, and the column was washed with different concentrations ofNaCl from 0.15 to 1 M. The eluted fractions were collected and, alongwith the flow-through fractions, were subjected to Western blotanalysis.

Solid Phase Binding Assay—96-Well enzyme-linked immunosorbent assayplates were coated overnight at 4° C. with different ECM components. Thecomponents were fibronectin, laminin, type I and IV collagens, Matrigel(20 μg/ml, Becton Dickinson), vitronectin (10 μg/ml, Sigma), fibrinogen(20 μg/ml, Sigma), heparin, chondroitin sulfate, hyaluronic acid (200μg/ml, Sigma), and the whole ECM extracts derived from LLC cells (100μg/ml). The coated plates were washed and blocked with 0.5% bovine serumalbumin. The affinity-purified Ang-1v5/His and Ang-2v5/His were addedinto the coated plates (100 ng/ml) for overnight incubation at 4° C.After extensive washing, the bound Ang-1 and Ang-2 were detected. Theassays were performed in triplicate.

Tie-2 Tyrosine Phosphorylation Assay—LLC carcinoma cells expressingAng-1 or Ang-2 were cultured in 100-mm dishes until confluence and werelifted from the dishes as described. The dishes containing the ECMcomponents deposited by the cultured cells were used immediately.

HUVECs (ATCC) were cultured until subconfluence, and switched into theserum-free medium overnight. The cells were then lifted with 0.53 mMEDTA in Hanks' balanced buffer (Life Technologies, Inc.) and washed.1×106 HUVECs were seeded onto a plastic dish or one of the ECM-coateddishes freshly generated as described above. They were cultured at 37°C. in serum-free medium with or without soluble Ang-1 (200 ng/ml) orTie-2-Fc fusion proteins (2 μg/ml) for 30 min. The cells were then lysedat 4° C. with the lysis buffer (50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1%Triton X-100, 2 mM EDTA, 2 mM sodium orthovanadate, 2 mM sodiumfluoride, 2 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 1 mMpepstatin A, and 10 μg/ml aprotinin). Tie-2 proteins wereimmunoprecipitated using anti-Tie-2 polyclonal antibody (Santa CruzBiotechnology). The immunoprecipitated proteins were subjected Westernblot analysis using anti-phosphotyrosine antibody (Y20, Calbiochem).

Results

Angiopoietin-1, but not Angiopoietin-2, Binds to the ExtracellularMatrix—In the process of studying the role of angiopoietins in tumorangiogenesis, we experienced difficulty obtaining the transfected tumorcells that secrete high levels of Ang-1, whereas the transfectants thatsecrete high levels of Ang-2 were easily obtained. One possible reasonfor the phenomena is that Ang-1 and Ang-2 may associate with the ECMdifferently. To investigate that, we generated the stably transfectedLewis lung carcinoma (LLC) and TA3 murine mammary carcinoma (TA3) cellsexpressing v5 epitope-tagged Ang-1 or Ang-2 (FIG. 1, A, a and b). Undernon-reducing conditions both Ang-1 and Ang-2 tend to aggregate with eachother to form dimers and oligomers (FIGS. 1, A and B). The patterns ofthe aggregation are distinct between Ang-1 and Ang-2. Ang-1 tends toform higher order oligomers (FIG. 1A), whereas Ang-2 forms dimers,trimers, and oligomers. The molecular weight of the monomer of Ang-1 andAng-2 is ˜70 kDa. Because of the C-terminal v5 and 6× histidine epitopetags, Ang-1v5 and Ang-2v5 migrate a little slower.

The aggregation of Ang-1 and Ang-2 is sensitive to the reducing agents,such as -mercaptoethanol. After boiling the protein samples in 1×Laemmli SDS sample buffer containing 5%-mercaptoethanol, the aggregatedAng-1 and Ang-2 are dissociated into monomers (data not shown). It wasnoted that the amount of the secreted Ang-1 is often lower than that ofAng-2 derived from LLC and TA3 transfectants (FIGS. 1A, a and b). Toexamine the possibility that the lack of Ang-1 secretion is due toincorporation of Ang-1 into the ECM of the tumor cells, thetransfectants expressing Ang-1 and Ang-2 were grown until confluence,and the cells were then detached from the culture dishes by the EDTAtreatment, which is a standard procedure to release cultured cells andleave the ECM components behind on the culture dishes (38). Theremaining ECM components were then extracted from the culture dishes by1×SDS Laemmli buffer with or without -mercaptoethanol. Western blotanalyses were performed using anti-v5 monoclonal antibody to detectv5-tagged Ang-1 or Ang-2. The results indicated that Ang-1 is present inthe ECM fraction of the transfected tumor cells, whereas Ang-2 is absent(FIG. 1B). The ECM-associated Ang-1 is highly aggregated to formoligomers, and no monomer is detected (FIG. 1B, lanes 1 and 2, and 6 and7). The aggregated Ang-1 oligomers were dissociated into the monomers bythe treatment with -mercaptoethanol (FIG. 1C), which indicates the roleof the cysteine residues in the aggregations. Thus, we have establishedthat Ang-1, but not Ang-2, is incorporated into the ECM of thosecarcinoma cells.

To confirm that the endogenously expressed Ang-1 is associated with theECM as the transfected Ang-1, the polyclonal anti-Ang-1 antibody (C19,Santa Cruz Biotechnology) was first purified through anAng-1v5-conjugated protein A affinity column. The purified antibody wasused in Western blot analysis of the ECM components derived from HUVECs.

The results indicated that Ang-1 produced by HUVECs is incorporated intothe ECM (data not shown). Due to the superior quality of the anti-v5monoclonal antibody compared with that of the polyclonal anti-Ang-1antibody, even after the affinity purification (Santa CruzBiotechnology), most of the following experiments were performed usingthe transfected tumor cells expressing v5-tagged Ang-1 and Ang-2.

The Biochemical Characters of the ECM-associated Ang-1—The strength ofthe ECM association of Ang-1 was tested by extracting the cell-free ECMdeposited on the culture dishes by LLC cells expressing Ang-1v5 withdifferent concentrations of sodium chloride (NaCl) and deoxycholate(DOC). The insoluble materials after the extractions were solubilizedwith 1×SDS Laemmli buffer and subjected to Western blot analysis. Asshown in FIG. 2, most of the ECM-incorporated Ang-1 resisted 0.15 M NaClextraction (FIG. 2, lane 2); a fair amount of Ang-1 remains in the ECMafter 1 M NaCl extraction (FIG. 2, lane 4), and a fraction of Ang-1 isin the ECM fraction even after 1% DOC extraction (FIG. 2, lane 6), whichindicated a strong ECM association and implicated a gradual assemblyprocess of Ang-1 into the ECM (FIG. 2).

The Binding Affinity of Ang-1 to Different ECM Components—The C terminusof Ang-1 shares the sequence homology with fibrinogen, which binds toheparin. To investigate whether Ang-1 binds to the ECM via itsinteraction with the sulfated glycosaminoglycans, the purifiedAng-1v5/His (FIG. 3A, lane 1) was applied to a heparin-Sepharose CL-6Baffinity column (Amersham Pharmacia Biotech). The unbound flow-throughwas collected, and the column was washed with 0.15, 0.3, 0.5, 1 M NaCl(FIG. 3A, lanes 3-6). The eluted fractions were collected, and alongwith the flow-through fraction, they were subjected to the Western blotanalysis by using anti-v5 monoclonal antibody to detect the presence ofAng-1v5/His in each fraction. The results indicated that Ang-1v5/Hisdoes not bind to the heparin affinity column and was fully recovered inthe flow-through fraction (FIG. 3A, lane 2). To confirm the abovefinding and avoid the possibility that the purified soluble Ang-1v5/Hismay be modified and different from the ECM-associated Ang-1 and therebyunable to bind to heparin, soluble sulfated glycosaminoglycans, heparin,and chondroitin sulfate (200 μg/ml) were added into serum-free cellculture medium (SFM) of LLC carcinoma cells expressing Ang-1. After 2,12, and 24 h of incubation, the SFM and ECM fractions were collected andanalyzed. The Western blot results indicated that neither heparin norchondroitin sulfate releases Ang-1 from the ECM of the transfected LLCcells (FIG. 3B, and data not shown).

In an attempt to identify the ECM component(s) that bind(s) to Ang-1,solid phase binding assays were performed to assess the binding affinityof Ang-1 to several ECM components. The assays were performed intriplicate, and the results are listed in the FIG. 4. The purifiedAng-1v5 binds to whole ECM extracts derived from LLC carcinoma cellswith high affinity (FIG. 4, column 8). The weak bindings to Matrigel,fibrinogen, and vitronectin were observed, which can't account entirelyfor the high affinity binding between Ang-1 and the ECM extracts. Ang-1displayed no affinity to fibronectin, laminin, collagen type I and typeIV, heparin, chondroitin sulfate, and hyaluronic acid (FIG. 4). Thisresult offered the possibility that Ang-1 binds to the ECM via anunidentified ECM protein(s).

Immunocytochemistry Studies Revealed a Distinct ECM Distribution Patternof Angiopoietin-1—The distribution patterns of Ang-1 and Ang-2 wereinvestigated and compared with fibronectin, laminin, and type I and IVcollagens by performing immunocytochemistry on LLC carcinoma cellsexpressing Ang-1v5 or Ang-2v5 using anti-v5 antibody or antibodiesagainst the appropriate ECM proteins. The cell-free ECM deposited by LLCcells expressing Ang-1 or Ang-2 was also examined. Theimmunocytochemistry studies uncovered a distinct Ang-1 distributionpattern in the ECM of LLC carcinoma cells, which is different from thedistribution of fibronectin, laminin, and types I and IV collagen (FIGS.5, A and B). Ang-1 is incorporated into the ECM as small granule-likedepositions, which are more or less evenly distributed beneath the cells(FIGS. 5A, a and b). Some cell-free spaces, which are left behind by themigrating cells, are positive for the similar Ang-1 depositions (FIG.5A, a, arrow). The distribution pattern of Ang-1 was preserved evenafter the cells were lifted by the treatment of EDTA (FIG. 5A, b), whichindicated that instead of loosely binding to the tumor cells, thoseAng-1 proteins are incorporated into the ECM of the tumor cells. On thecontra, there is no trace of Ang-2 in the cell-free ECM deposited by thecells expressing Ang-2 (FIG. 5A, d). Ang-2 was only detected in thecytoplasm of the transfected cells, which presumably reflects thepresence of Ang-2 in the secretory pathway of the cells (FIG. 5A, c,arrowheads).

Angiopoietin-1 Is Released in the Response to Phorbol 12-Myristate13-Acetate (PMA)—The association of Ang-1 with the ECM of tumor cellsled us to explore the regulatory mechanisms of its incorporation andreleasing, which may modulate its activity. The confluent LLC carcinomacells expressing Ang-1 were cultured overnight in the presence ofdifferent growth factors or PMA in serum-free cell culture medium. Afterreleasing the cells from the culture dishes, the remaining ECMcomponents were extracted with 1×SDS Laemmli buffer and subjected toWestern blot analysis using anti-v5 antibody. The results indicated thatPMA stimulates the releasing of the ECM-associated Ang-1 (FIG. 6, lane2). Transforming growth factor-1 (TGF-1) promotes slightly theincorporation of Ang-1 into the ECM, which may reflect the positiveeffect of TGF-1 on synthesis of the ECM components (FIG. 6, lane 3).

The ECM-associated Ang-1 Does Not Bind to Tie-2-Fc Fusion Protein—Todetermine whether the ECM-associated Ang-1 proteins bind to Tie-2-Fcfusion proteins, the confluent LLC cells expressing Ang-1 or the ECMdeposited by the LLC cells expressing Ang-1 were fixed and incubatedwith anti-v5 antibody or the purified Tie-2-Fc fusion proteins. Anti-v5antibody and FITC-conjugated rabbit anti-mouse antibody (Sigma) revealedthat many Ang-1 proteins were deposited into the ECM of the tumor cells(FIGS. 7B, a and b); however, Ang-1 proteins in the ECM exhibited noaffinity to Tie-2-Fc fusion proteins (FIGS. 7B, c and d). In order toeliminate the possibility that the fixation procedure could affect thebinding between the ECM-associated Ang-1 and Tie-2-Fc fusion protein,Tie-2-Fc fusion proteins were directly added into the cultured LLCcarcinoma cells expressing Ang-1 or into the ECM freshly derived fromthe LLC cells expressing Ang-1. No binding was detected in both cases(data not shown). Those ECM-associated Ang-1 proteins were extracted by2 M urea/Tris-HCl buffer (pH 7.4), dialyzed in PBS, andimmunoprecipitated using Tie-2-Fc fusion proteins and protein A beads.The precipitated proteins were subjected to Western blot analysis usinganti-v5 antibody. Ang-1 proteins were found to be precipitated byTie-2-Fc fusion proteins (FIG. 7A, lane 2). Together, those resultssuggested that after their secretion from the tumor cells, Ang-1proteins are incorporated into the ECM, and their Tie-2-binding sitesare no longer accessible; upon the release from the ECM, the solubilizedAng-1 proteins regained their ability to bind to Tie-2. Underphysiologic conditions, this may serve as an efficient regulatorymechanism for Ang-1 activity.

Tie-2 Phosphorylation Is Achieved by Adhering HUVECs to the ECMContaining Ang-1 Proteins—To study whether the ECM-associated Ang-1plays a role in angiogenesis, the ability of the ECM-associated Ang-1 topromote Tie-2 receptor phosphorylation was evaluated using HUVECs. Thesubconfluent HUVECs were serum-starved for about 14 h and then liftedfrom the culture dishes. 1×106 cells were seeded onto a plastic culturedish with (FIG. 8, lane 3) or without (FIG. 8, lane 1) purified solubleAng-1 (200 ng/ml) or the culture dish containing the ECM componentsdeposited by the confluent LLC cells expressing Ang-1 (FIG. 8, lane 4)or Ang-2 (FIG. 8, lane 2). The HUVECs were incubated on those cellculture dishes for 30 ml and then lysed at 4° C. with the lysis buffer.The amount of the Tie-2 proteins in the lysates was determined byWestern blot analysis of 50 μg of proteins from each lysate usinganti-Tie-2 antibody (C-20, Santa Cruz Biotechnology, FIG. 8B). Theresults indicated that the endothelial cells used in the experimentsexpress a similar amount of Tie-2 receptors (FIG. 8B). Thephosphorylated Tie-2 proteins in the lysates were assessed by performingimmunoprecipitation using anti-Tie-2 polyclonal antibody (C-20, SantaCruz Biotechnology) and analyzing the precipitated proteins on Westernblot using anti-phosphotyrosine antibody (Y20, Calbiochem). The resultsindicated that Tie-2 receptors on HUVECs are phosphorylated by Ang-1derived from the ECM, and the phosphorylation is inhibited by theaddition of the excess amount of Tie-2-Fc fusion proteins (2 μg, FIG. 8,lane 5).

To assess whether HUVECs release Ang-1 proteins from the ECM, HUVECscells (1×106 cells/100-mm dish) or the serum-free culture media (SFM)alone were placed on the ECM deposited by LLC cells expressing Ang-1 for30 min. The cell culture dishes with or without HUVECs were treated with5 mM EDTA in PBS for 5 min to release the adhering cells. The remainingECM materials on the culture dishes were extracted and subjected toWestern blot analysis, and the results indicated that the reduced amountof Ang-1 is retained in the ECM after the adhesion of HUVECs comparedwith that retained in the ECM incubated with SFM alone (data not shown).Thus, HUVECs promote the release of Ang-1 from the ECM.

The Domain That Mediates the ECM Binding of Ang-1 Is Mapped to ItsLinker Peptide Region—To determine which domain of Ang-1 mediates itsECM association, several expression constructs were made as indicated inFIG. 9A. They were the coiled-coil domain, the coiled-coil plus linkerpeptide region, and the FHD of Ang-1. The cDNA sequence encoding thesignal peptide of Ang-1 was constructed into the N terminus of the abovecDNA fragments so those fragments can be secreted properly (FIG. 9A).The full-length Ang-1 and -2 and the fragments of Ang-1, which containC-terminal v5 epitope tags, were used to transfect COS-7 cells. 72 hafter the transient transfection, the cell culture supernatants and theECM materials derived from the transfected cells were either collectedor extracted from the cell culture dishes and subjected to Western blotanalyses using anti-v5 antibody to determine the distribution patternsof Ang-1, Ang-2, and the different fragments of Ang-1. The resultsindicated that the linker peptide region of Ang-1 between thecoiled-coil and the fibrinogen-like domains, which contains 27 aminoacids, is mainly responsible for the ECM association of Ang-1 (FIGS. 9,B and C, lanes 5 and 6). A weak interaction between the coiled-coildomain of Ang-1 and the ECM was also detected (FIGS. 9, B and C, lanes 7and 8).

To investigate whether the ECM binding domain in the linker peptideregion of Ang-1 is also present in Ang-2, we first compared the sequencehomology between the domains of Ang-1 and Ang-2. We found that thepercentages of the identical amino acids are 59, 19, and 64%,respectively, in the coiled-coil domain, the linker peptide region, andthe fibrinogen-like domains of Ang-1 and Ang-2. No significant homologyin the linker peptide region implied that the ECM binding domain in thisregion of Ang-1 is likely absent in the Ang-2 molecule.

To confirm that the absence of the ECM binding of Ang-2 is due to thelack of the ECM-binding site(s) in the molecule, but not the blockage ofthe binding site(s) by possible steric restraints in the full-lengthAng-2 molecule, we made three additional deletions of Ang-2, which aresimilar to those of Ang-1 deletions (FIG. 9A). They are the coiled-coildomain, the coiled-coil plus the linker peptide region, and thefibrinogen-like domain of Ang-2. All the deletion constructs contain theN-terminal signal peptides of Ang-2 for their proper expression andsecretion and the C-terminal v5 epitope tags derived from thepEF/6/v5-His expression vectors for their detection. After confirmingthe authenticities of the expression constructs by DNA sequencing, theywere used to transfect COS-7 cells. After the transient transfection,the distributions of Ang-2 fragments in the cell culture media and theECM fractions were examined by Western blot analysis. Our resultsindicated that all three fragments of Ang-2, the coiled-coil domain, thecoiled-coil plus the linker peptide region, and the fibrinogen-likedomain, are secreted as the full-length Ang-2; none of them associateswith the ECM of the transfected COS-7 (data not shown). This resultindicated clearly that unlike Ang-1, Ang-2 does not contain the ECMbinding domain.

Discussion

Studies have shown that angiopoietin-1 and -2 are unique antagonists.Ang-2 blocks tyrosine phosphorylation of Tie-2 induced by Ang-1 anddisrupts angiogenesis in vivo. Ang-1 and -2 play different roles intumor angiogenesis. Ang-2 inhibits tumor angiogenesis by blockingrecruitment of smooth muscle cells to the newly formed blood vessels andcausing apoptosis to the endothelial cells.

Functional Significance of the ECM Association of Ang-1—In this study,we examined the potential relationship of Ang-1 and -2 with the ECM,which is known to modulate activities of many growth factors, includingVEGF and bFGF. Our results indicated that unlike Ang-2, Ang-1 issecreted and incorporated into and sequestered by the ECM. The differentECM association capacity enables those two antagonists to regulate localand distant angiogenesis differently. Ang-1 is most likely to regulateangiogenesis in the vicinity of its secretion sites, whereas Ang-2 islikely to diffuse through interstitial tissues and blood vessels to thedistant organs.

The ability to distribute Ang-1 and -2 to local environment and/ordistant sites may play an important role in regulating tumor dormancy insome situations. Both Ang-1 and -2 are expressed by endothelial andtumor cells. During the growth of primary tumors, balanced expression oflocal Ang-1 and -2 and expression of other pro-angiogenic factors, suchas VEGF, may ensure tumor angiogenesis and growth in the primary sites.When Tie-2 receptors on local endothelial cells are saturated, it islikely that the excess amount of Ang-1 produced by the primary tumorswould be incorporated into and sequestered by the surrounding ECM,whereas the excessive amount of Ang-2 may diffuse to distant organs. Themicrometastases seeded and developed in the distant organs, such as lungand liver, have microenvironments that contain pro- and anti-angiogenicfactors produced by local micrometastatic tumors and surrounding hoststromal tissues and are more or less similar to the microenvironments ofprimary tumors. However, on top of the microenvironments, Ang-2, notAng-1, produced by the predominant primary tumors, which is stable andhas a long half-life (data not shown), could travel to the distantorgans and change the balance in favor of inhibiting tumor angiogenesis,which may contribute to the dormancy observed in some secondary tumors.

The ECM Association of Ang-1 Provides a Different Regulatory Mechanismfor the Availability of Ang-1—Ang-1 is wildly expressed both in embryoand in adult and exhibits a more uniform expression pattern comparedwith that of Ang-2 Ang-2 has a wild expression pattern in embryo.However, in adult tissues, its expression is restricted in the tissueswhere vascular remodeling is ongoing, such as ovary, uterus, andplacenta. Ang-2 is up-regulated by hypoxia and several differentcytokines including VEGF and tumor necrosis factor, whereas theexpression of Ang-1 is not affected.

The association of Ang-1 with the ECM reported herein offered adifferent type of regulation of the availability of Ang-1. Instead ofregulating its production, as Ang-2, free Ang-1 in microenvironment canbe regulated by changing its ECM association status. The factors thatpromote or inhibit its ECM incorporation or releasing affect theinteraction between Ang-1 and Tie-2 receptor, sequential signaltransduction, and angiogenesis. This type of regulation provides aquicker response to the changes in the microenvironment without therequirement of mRNA and proteins syntheses. It is well established thatthe activities of some growth factors are regulated in that way,including TGF.

The Biochemical Characters of the ECM Association of Ang-1—An extensiveeffort was made to try to identify the ECM component(s) that mediate(s)the ECM association of Ang-1. Matrigel, fibrinogen, and to a less extentvitronectin exhibited weak affinities to Ang-1, which is much lower thanthat of unpurified ECM extracts derived from LLC cells and cannotaccount entirely for the ECM association of Ang-1 (FIG. 4). Theimmunocytochemistry studios indicated that the ECM distribution patternof Ang-1 is unique and unlike that of fibronectin, laminin, and collagentypes I and IV (FIG. 5).

To determine the effect of the ECM association on the function of Ang-1,we assessed whether Tie-2-Fc fusion protein hinds to the ECM-associatedAng-1. The result indicated that the ECM-associated Ang-1 is notaccessible for Tie-2, and the ECM serves as a storage and sequestrationsite for Ang-1. The incubation of HUVECs on the ECM containing Ang-1induced the release of Ang-1 from the ECM and tyrosine phosphorylationof Tie-2, which indicated that HUVECs cells are able to respond to Ang-1originally sequestered in the ECM (FIG. 8). This may reflect the in vivosituations and indicate that activity of Ang-1 is restricted in localenvironment and regulated more tightly than that of Ang-2.

The Domain of Ang-1 That Mediates Its ECM Association Is the LinkerPeptide Region—The deletion analysis indicated that the linker peptideregion of Ang-1 between the coiled-coil domain and the FHD mediates theECM association of Ang-1 (FIG. 9). The identification of the ECMassociation domain of Ang-1 will allow us to study the importance of theECM association on Ang-1 function in the future.

In summary, we have discovered that Ang-1 is incorporated into the ECMafter its secretion, whereas Ang-2 is secreted and does not associatewith the ECM. The domain that mediates the ECM association of Ang-1 ismapped to the linker peptide region between the coiled-coil and thefibrinogen homology domains. The different ECM association capacity ofAng-1 and Ang-2 offers a possible mechanism for the distinct regulationsof local and distant tumor angiogenesis by two antagonistic factors.

ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; Ang-1,angiopoietin-1; Ang-2, angiopoietin-2; TA3, TA3 murine mammarycarcinoma; LLC, Lewis lung carcinoma; HUVEC, human umbilical veinendothelial cell; FHD, fibrinogen homology domain; PBS,phosphate-buffered saline solution; BSA, bovine serum albumin; PCR,polymerase chain reaction; RT-PCR, reverse transcriptase-polymerasechain reaction; FITC, fluorescein isothiocyanate; DOC, deoxycholate;SFM, serum-free medium; PMA, phorbol 12-myristate 13-acetate; bFGF,basic fibroblast growth factor; TGF-, transforming growth factor-; VEGF,vascular endothelial growth factor.

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1. A pharmaceutical composition comprising a) a pharmaceuticallyacceptable carrier and b) a therapeutically effective amount of a vectorcomprising a nucleic acid molecule that comprises the nucleotidesequence that encodes an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO:2 or a homologous peptidethereof.
 2. The pharmaceutical composition of claim 1 further comprisinga therapeutically effective amount of an ECM-binding fragment of Ang-1protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a homologouspeptide thereof.
 3. The pharmaceutical composition of claim 1 comprisinga vector comprising a nucleic acid molecule that comprises thenucleotide sequence that encodes an ECM-binding fragment of Ang-1protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2.
 4. Thepharmaceutical composition of claim 3 comprising wherein said vector isa viral vector.
 5. The pharmaceutical composition of claim 3 comprisingwherein said vector is a DNA plasmid.
 6. A method of treating anindividual suspected of having coronary artery disease, vascular diseaseor a condition involving ischemia comprising the step of administeringto said individual a pharmaceutical composition comprising a) apharmaceutically acceptable carrier and b) therapeutically effectiveamount of a vector comprising a nucleic acid molecule that comprises thenucleotide sequence that encodes an ECM-binding fragment of Ang-1protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a homologouspeptide thereof.
 7. The method of claim 6 wherein said pharmaceuticalcomposition further comprises a therapeutically effective amount of anECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1 and/orSEQ ID NO:2 or a homologous peptide thereof.
 8. The method of claim 6wherein said pharmaceutical composition comprises a vector comprising anucleic acid molecule that comprises the nucleotide sequence thatencodes an ECM-binding fragment of Ang-1 protein that comprises SEQ IDNO:1 and/or SEQ ID NO:2.
 9. A method of promoting angiogenesis,endothelial survival and maintaining vascular integrity in an individualcomprising the step of administering to said individual a pharmaceuticalcomposition comprising a) a pharmaceutically acceptable carrier and b)therapeutically effective amount of a vector comprising a nucleic acidmolecule that comprises the nucleotide sequence that encodes anECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1 and/orSEQ ID NO:2 or a homologous peptide thereof.
 10. The method of claim 9wherein said pharmaceutical composition further comprises atherapeutically effective amount of an ECM-binding fragment of Ang-1protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a homologouspeptide thereof.
 11. The method of claim 9 wherein said pharmaceuticalcomposition comprises a vector comprising a nucleic acid molecule thatcomprises the nucleotide sequence that encodes an ECM-binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2.
 12. Amethod of identifying compounds that modulates binding of Ang-1 to ECMcomprising performing a test assay that comprises the steps of: a)contacting a protein that comprises at least an ECM-binding fragment ofAng-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with ECMmaterial in the presence of a test compound; b) measuring the level ofbinding of said protein that comprises at least an ECM-binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with saidECM; and c) comparing said level with the level of binding of proteinthat comprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO: and/or SEQ ID NO:2 with ECM material in the absenceof said test compound; wherein when the level of binding of said proteinthat comprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO:2 with said ECM in the presenceof said test compound is less than the level of binding of said proteinthat comprises at least an ECM-binding fragment of Ang-1 protein thatcomprises SEQ ID NO:1 and/or SEQ ID NO:2 with said ECM in the absence ofsaid test compound results indicate that the test compound modulatesbinding of Ang-1 to ECM by inhibiting said binding and when the level ofbinding of said protein that comprises at least an ECM-binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with saidECM in the presence of said test compound is more than the level ofbinding of said protein that comprises at least an ECM-binding fragmentof Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with saidECM in the absence of said test compound results indicate that the testcompound modulates binding of Ang-1 to ECM by enhancing said binding.13. The method of claim 12 wherein the protein is Ang-1 protein.
 14. Themethod of claim 12 wherein the protein is a fragment of Ang-1 protein.15. The method of claim 12 wherein the protein comprises a detectablelabel.
 16. The method of claim 15 wherein the level of binding ismeasured by measuring the amount of detectable label present on said ECMafter removing unbound protein from said ECM material.
 17. The method ofclaim 12 wherein the ECM material is produced by culturing cells on asubstrate for a sufficient time for said cells to produce ECM materialon said substrate and removing said cells from said substrate withoutremoving said ECM material.
 18. The method of claim 17 wherein saidcells are Lewis lung carcinoma cells or TA3 murine mammary carcinomacells.
 19. The method of claim 12 wherein said method comprisingmultiple test assays which are identical except that the amount of testcompound used differs.
 20. The method of claim 12 the level of bindingof protein that comprises at least an ECM-binding fragment of Ang-1protein that comprises SEQ ID NO; I and/or SEQ ID NO:2 with ECM materialin the absence of said test compound is determined by performing acontrol assay wherein said control assay comprises the steps of a)contacting a protein that comprises at least an ECM-binding fragment ofAng-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with ECMmaterial in the absence of a test compound; b) measuring the level ofbinding of said protein that comprises at least an ECM-binding fragmentof Ang-1 protein that comprises SEQ ED NO:1 and/or SEQ ID NO:2 with saidECM.
 21. A pharmaceutical composition comprising a) a pharmaceuticallyacceptable carrier and b) a therapeutically effective amount of Ang-2protein and/or a vector comprising a nucleic acid molecule thatcomprises the nucleotide coding sequence of Ang-2.
 22. Thepharmaceutical composition of claim 21 comprising a therapeuticallyeffective amount of Ang-2 protein.
 23. The pharmaceutical composition ofclaim 21 comprising a vector comprising a nucleic acid molecule thatcomprises the nucleotide coding sequence of Ang-2.
 24. Thepharmaceutical composition of claim 23 comprising wherein said vector isa viral vector.
 25. The pharmaceutical composition of claim 23comprising wherein said vector is a DNA plasmid.
 26. A method oftreating an individual suspected of having cancer comprising the step ofadministering to said individual a pharmaceutical composition comprisinga) a pharmaceutically acceptable carrier and b) a therapeuticallyeffective amount of Ang-2 protein and/or a vector comprising a nucleicacid molecule that comprises the nucleotide coding sequence of Ang-2.27. The method of claim 26 wherein said pharmaceutical compositioncomprises a therapeutically effective amount of Ang-2 protein.
 28. Themethod of claim 26 wherein said pharmaceutical composition comprises avector comprising a nucleic acid molecule that comprises the nucleotidecoding sequence of Ang-2.
 29. The method of claim 28 wherein said vectoris a viral vector.
 30. The method of claim 28 wherein said vector is aDNA plasmid.